Preparation of Affinity Column

Preparation of Affinity Column

SEK 5/3/95

1.Add 222.2μl 1M MOPS,pH 7.5 to 2ml of 10mg of CREBtide (0.1M MOPS pH 7.5 final concentration).

2.Read OD₂₀₅,OD₂₈₀ (using 0.1M MOPS buffer as blank)

3.Affigel-10 is stored frozen at < -70℃.Thaw at 4℃ for ~20min.

4.Filter 1-2ml on nylon (0.22uM poresize),using Buchner funnel and vacuum.Do not allow beads to dry.

5.Wash bed with 3x2ml dH₂O.

6.Scrape 0.5-1ml into 15ml microfuge tube.

7.Add 2.22ml CREBtide ligand solution to tube.

Rock at 4℃ for 4hrs.

8.Transfer slurry to column.

9.Wash 2x500μl dH₂O.

10.Collect eluate and save at -20℃.Read OD.

11.Wash column:

2x5ml cold PBS

1x5ml PBS/0.02% NaN3 (1μl NaN3 in5ml PBS)

12.Store at 4℃ upright in beaker in PBS/NaN³⁺ parafilm.